Synthesis of streptamine



Patented Mar. 25, 1952 SYNTHESIS OF STREPTAMINE Melville Lawrence Wolfram, Columbus, Ohio, and Stephen Munro Olin, Elkhart, Ind., assignors to The Ohio State University Research Foundation, Columbus, Ohio, a corporation of Ohio No Drawing. Application April 19, 1949, Serial No. 88,499

13 Claims.

l tHH Thus it is seen that the molecule of the antibiotic streptomycin consists of three moieties, N- methyl-glucosamine, 3-C-formyl-5-desoxy-L- aldopentofuranoside and a meso-diguanidineinositol.

Streptidine was first isolated by the acid hydrolysis of streptomycin hydrochloride as the crystalline sulfate and picrate. (Science, 103, 53 (1946)).

Strong alkaline hydrolysis of streptidine would y'ieldfour moles of ammonia and two moles of carbon dioxide with the production of a compouncl having the following formula:

; anon cfion 011N112 ('JHOH HOE This compound 1,3-diamino-2,4=,5,6-tetrahydroxycyclohexane will hereinafter be designated streptamine (J. Fried, G. A. Boyack and 0. Win 0 tersteiner, J. Biol. Chem, 162, 391 (1946) Streptamine forms a hexaacetate which crystallizes in two forms: a chloroform-insoluble fraction crystallizing in long needles of M. P. 343-345 with a characteristic transition point at 250 to longer needles, and a chloroform-soluble fraction which possesses the same characteristic transition point and melting point as the chloroform-insoluble form. (R. L. Peck, C. E. Hoffman, Jr., Elizabeth W. Peel, R. P. Graber, F. W. Holly, R. Mozingo and K. Folkers, J. Am. Chem. Soc. 68, 776 (1946)).

The present invention involves a novel process for the preparation of streptamine comprising the following steps:

1. Reacting N-acetyl-D-glucosamine with ethyl mercaptan to yield crude N -acetyl-D-gluosamine diethyl thioacetyl. This may be purified by the preparation of pentacetyl D-glucosamine diethyl thioacetal, chromotographic separation, and subsequent de-O-acetylation to yield a pure N- acetyl-D-glucosamine diethyl thioacetal.

2. Reacting N-acetyl-D-glucosamine diethyl thioacetal with mercuric chloride and mercuric oxide, to yield ethyl 2-desoxy-2-aeetamido-a-D- glucothiofuranoside.

3. Reacting ethyl-2-desoXy-Z-acetamide-a-D- glucothiofuranoside with lead tetraacetate, and treatment of the resulting product with nitromethane, neutralizing and crystallizing the product, a mixture of ethyl 2,6-didesoXy-2-acetamido' 6-nitro-a-D-glucothiofuranoside and ethyl 2,6- didesoxy-2-acetamido-6- nitro ,8 L idothiofuranoside. These products are then separated and the higher melting form is demercaptolated and condensed intramolecularly, acidified, hydrogenated, then acetylated to streptamine hexaacetate.

4. Streptamine hexaacetate is converted to streptamine by hydrolysis.

Step1 Step 2 Step3 CHO HJJNHAC HQ H H OH E1 1 OH EtSH Oone.HC1

HC(SEt)i HC(SE1,)2

HONHAc H NHAc HO H AcO H H OH H A0 H on OAe (J HzoH HQOAC HSEt H NHAc HO H H O H( OH UHiOH lPMOAcM HLSEt H NHAc HO H \/ HaONa H0 H HO OH H NHAc H NHaCl 11 H Pyridine l I OAc H I II EIACHNI OAcH 1 OAe H OAcH I \17 I A00 AeO 1 OAc H NHAe H NHAe Hexaaeetylstreptamine I NaOH NHIAc Isomer (probable configuration) H NHz by weight and all temperatures being by cen tigrade unless otherwise indicated.

Step 4 EXAMPLE 1 The preparation of pentaacetyl D-glucosamz'ne dieth'yl thioacetal N-acetyl-D-glucosamine (29 g.) was dissolved in concentrated hydrochloric acid cc.) at 0 and ethyl mercaptan (120 cc.) was added. The reaction mixture was stirred vigorously at 0 for twenty hours and then neutralized in the cold with concentrated ammonium hydroxide. The diethyl mercaptan layer was drawn off and discarded and the aqueous solution concentrated at reduced pressure. The resulting solids were dried by dissolving in absolute ethanol and concentrating at reduced pressure. An acetic anhydride-pyridine mixture cc. of two parts acetic anhydride, one part pyridine) was added to the dried solid and allowed to stand at room temperature for eighteen hours. The acetylation mixture was poured onto ice water (700 cc.) and extracted with four portions of chloroform ('75 Calculated Found C, 48. 47 C, 47. 95 47. 87 H, 6.71 H, 6.83 6.75 S, 12.93 8, 12.50

The yield of the acetylated mercaptal was 40% of the theoretical and numerous efiorts to increase the yield ,were fruitless. However, this was due to the poor crystallizing properties of the acetylated compound. By deacetylation of the sirup, yields up to 80% of the theoretical were obtained. Reacetylation of the diethyl thioacetal yielded a compound with the same constants as above and analyses of the same order of accuracy.

EXAMPLE 2 The preparation of N-acetyZ-D-glucosdmtne diethyl thioacetal (Z-desomy-Z-acetamido-D-glucose diethyl thz'oacetal) filtered. Anhydrous ether was added to the filtr-ate until the solution became cloudy. After standing overnight in the icebox, 1.5 g. of crystalline material was removed by filtration; M. P. 127-129, spec. rot. -23.4 (c 4, methanol, D-line, 23). After two recrystallizations the constants were as follows: M. P. 130-131,.spec. rot. -35.0 (c 4, methanol, D-line, 23). Further recrystallizations did not alter these constants.

An.aZysis.--Calculated for C12H25O5S2N:

Calculated Found EXAMPLE 3 The preparation of ethyl Z-desowy-Z-acetamidooc-D-gZMCOi'hiOflLTdflOSidC Yellow mercuric oxide was prepared from mercuric chloride (5 g.) and sodium hydroxide (2 g.)

6 according to the method of Green and mean. N-acetyl-D-glucosamine diethyl thioacetal (4.65 g. in 60 cc. of water) and the Washed oxide suspension were combined. Mercuric chloride (2.01 g.) dissolved in water (150 cc.) was added dropwise over a period of twenty minutes under vi orous mechanical stirring. At the end of the addition the reaction mixture was stirred for fifteen minutes. Pyridine (3 cc.) was added and the solution was filtered through a precoat of Celite. The filtrate was concentrated at reduced pressure and the resulting sirup was crystallized from ethanol, ether and one drop of py idine, yield 2.01 g.; M. P. 116-118, spec. rot. +1530 (c 3, water, D-line, 23) Three recrystallizations from the same solvents yielded 1.06 g. (30% of the theoretical) M. P. 1l9-12l, spec. rot. +170 (c 3, water, D-line, 22).

Analysis-Calculated for CmHmOsNS:

The preparation of ethyl Z-desoxy-Z-aminotetraacetyl-a-D-gluoothiofuranosikie N-acetyl D glucosamine diethyl thioacetal (10.0 g.) was treated as previously in the above thiofuranoside preparation. The resulting sirup w-as acetylated by dissolving in pyridine (60 cc.) and then adding acetic anhydride (60 cc.). The acetylation mixture was allowed to stand at room temperature for eighteen hours and then poured into ice water. The aqueous solution was extracted with four portions of chloroform cc.). The extract was washed with water, saturated aqueous sodium bicarbonate solution and finally with water. The dried chloroform solution was concentrated to a thin sirup and taken up in absolute ethanol. The solvents were removed at reduced pressure whereupon the concentratecrystallized; M. P. -90. Recrystallization from aqueous ethanol yielded a compound (2.51 g.) of M. P. 119-122", spec. rot. +123 (c 4, chloroform, D-line, 23). Two subsequent recrystallizations yielded a product of M. P. 124.5-125.5, spec. rot. (c 4, chloroform, D-line, 23). Further recrystallizations did not alter these constants.

Analysis.Calculated for C16H2508NSZ Calculated Found C. 49.47 C, 49. 22 H, 6.49 H, 6.72 N, 3.61 N, 3.77 S, 8.25 S, 8.08

,ing point.

semen 7) EXAMPLE The preparation of ethyl Z-desoxy-Z-aminotetraacetyZ-B-D-glucothiofuranoside Calculated lpnhd C, 49. 47 (3, 19. 21 H, 6. 40 n, 0.59 N, 3.61 N, 3.46 s, 8.25 s, 8.10

EXAMPLE 6 I The preparation of ethyl 2,6 didesory 2 acetamido-fi-nitro a D glucothiofuranoside and ethyl Zfi-didesory 2 acetamido-S-nitro-fi-L- idothiofuranoside Ethyl 2,desoxy 2 acetamido-a-D-glucothiofuranoside (2.51 g.) was dissolved in. absolute methanol (100 cc.). The solvent was removed at reduced pressure and the resulting sirup was dissolved in methanol (3 cc.) and chloroform (5i) cc). Lead tetraacetate (4.43 g.) in chloroform (100 cc.) was added and the reaction mixture warmed to 50-55. After fifteen minutes ,the Slight remaining color was removed by the addition of ethylene glycol (one drop).' .The chloroform solution was cooled in an ice bath and the lead diacetate was removed by filtration; The chloroform solution was extracted six times'with cold water in 2 cc. portions. The water extracts were extracted with chloroform. The c'hl'orc'i-- ,1.

' Raney nickel catalyst (200 mg.) 1n Water (30 cc.).

form solutions were dried and concentrated at reduced pressure. The resulting s'irup was taken up in 20 cc. of 95% ethanol and'initromethane 00.). The solution was made basic to litmus with 2 N sodium methylatc (5 cc.)

and the reaction mixture was allowed to stand 100 cc. and extracted With cold water. The dried chloroform solution was concentrated underreduced pressure to a yellow crystalline residue (0.45 g.). The isomers were separated coarsely by fractional crystallization from methanol,

Calculated Found 0, 40. so 0, 40. 48 H, 6.16 H, 6.26 N, 9.52 N, 9. 23 A Calculated Found C, 40. C, 40. 64 40. 58 H, 6.16 H, 5.78 5. 99 N, 9.52 N, 9.48 9.45

EXAMPLE 7 The preparation of streptamz'ne hexaacetate The high melting form M. P. 190-193 (with decomposition) from the preparation of the ethyl 2,6-didesoxy-2-acetamide-fi-nitro-a-hexothiofuranoside was used in the initial preparation of streptamine hexaacetate. One hundred sixtyfive miligrams of this compound was dissolved in warm Water (40 cc.) and mercuric chloride mg.) in water (10 cc.) was added. The reaction mixture was allowed to stand at room temperature for two hours. The mercury mercapto chloridewas removed by filtration. Silver acetate .(20 mg.) was added to the filtrate and the reaction mixture was kept at room temperature overnight. After the silver chloride and excess silver acetate were removed by filtration, hydrogen sulfide was passed into the filtrate. The sulfides'were removed by filtration and the filtrate blown to a clear sirup. All efforts to obtain a crystalline material from this sirup failed. Eighty milligrams of the above sirup was dissolved in water (1 cc.) and 1.60 cc. of barium hydroxide (0.1944 N) was added. On standing twenty-four hours at room temperature the solution turned straw colored. Absolute ethanol (10 cc.) was added and a highly colored flocculent precipitate was removed by filtration. .The solvent was removed in a stream of air and efforts to obtain a crystalline barium salt failed. Fifty milligrams of the barium salt was dissolved in 0.1 N hydrochloric acid (4 cc.) and combined with Thesolution was hydrogenated at room tempera ture under one atmosphere pressure. The catal-y'st was removed at the centrifuge and washed with ethanol. The supernatant liquid and washings were blown to dryness and acetylated in acetic anhydride-pyridine mixture (3 cc. of 2:1) under gentle reflux for one hour. The reaction mixture was blown to dryness and extracted with warm chloroform. The residue from the'chloroform extraction was treated with warm absolute ethanol. The absolute ethanol solution was filtered and placed in the ice box: after twelve hours 4 mg. of needle clusters had formed. The needles were removed by filtration and X-ray diffraction pattern was taken of this material. This compound was shown to be identical with streptamine hexaacetate on the basis of this evidence. The compound also possessed a similar transition point and melting points as strepf tamine hexaacetate prepared from the degradation product of streptomycin; transition point 245-250, M. P. 345-348 (in sealed capillary). The chloroform solution was blown to dryness and elongated prisms formed which were insoluble in cold chloroform. The compound was removed from the chloroform solution by filtration, yield 5 mg. of M. P. 350-355 with decomposition. A powder X-ray diffraction pattern was taken of this material. It was shownonthis evidence to be neither the chloroform soluble or chloroform insoluble crystalline form of streptamine hexaacetate.

X-ray diffraction patterns Streptamine Hcxaacetate Strcptaminc Hcxaacctatc from Streptomycin from D-Glucosaminc Interplan ar Relative lnterplangr Relative Spacing, A. Intensity Spacing, A. Intensity EXAMPLE 8 1 gram of streptamine hexaacetate is refluxed with 100 m1. of 6 N sodium hydroxide for 48 hours. After neutralization streptamine is isolated as the slightly soluble sulfate salt.

It is to be understood that the above particu lar description is by way of illustration, and that changes, omissions, additions, substitutions and/or modifications may be made within the scope of the claims without departing from the spirit of the invention which is intended to be limited only as required by prior art.

We claim:

1. Process of preparing streptamine which comprises reacting N-acetyl-D-glucosamine with ethyl mercaptan to obtain N-acetyl-D-glucosamine diethyl thioacetal, reacting the N-acetyl-D-glucosamine diethyl thioacetal with mercuric chloride and mercuric oxide to obtain ethyl 2-desoxy- 2-acetamido-a-Dglucothiofuranoside, reacting the ethyl 2-desoxy-Z-acetamido-e-D-glucothiofuranoside with lead tetraacetate, treating the resulting product with nitromethane and crystallizing a mixture of ethyl 2,6-didesoxy-2-acetamido- 6-nitro-a-D-glucothiofuranoside and ethyl 2,6- didesoxy-2-acetamido-6-nitro-,B-L- idothiofuranoside, separating, and demercaptolating the highor melting form, intramolecularly condensing, hydrogenating and acetylating to streptamine hexaacetate, which is subsequently hydrolyzed to streptamine.

2. Process which comprises reacting N-acetyl- D-glucosamine With ethyl mercaptan to obtain N acetyl D glucosamine diethyl thioacetal, reacting the N acetyl D glucosaminc diethyl thioacetal with mercuric chloride and mercuric oxide to obtain ethyl 2-desoxy-2-acetamido-a- D-glucothiofuranoside, reacting the ethyl 2-desoxy 2 acetamido-a-D-glucothiofuranoside with lead tetraacetate, treating the resulting product with nitromethane and crystallizing a mixture of ethyl 2,6-didesoxy-2-acetamido-6-nitro-a-D 10 glucothiofuranoside and ethyl 2,6-didesoxy-2-acetamido-G-nitro/3-Lidothiofuranoside, separating, and demercaptolating the higher melting form, intramolecularly condensing, hydrogenating and acetylating to streptamine hexaacetate.

3. Process which comprises reacting N-acetyl- D-glucosamine with ethyl mercaptan to obtain N-acetyl-D-glucosamine diethyl thioacetal, reacting the N-acetyl-D-glucosamine diethyl thioacetal with mercuric chloride and mercuric oxide to obtain ethyl 2-desoxy-2-acetamido-a-D-gluco thiofuranoside, reacting the ethyl 2-desoXy-2- acetamido-a-Dglucothiofuranoside with lead tetraacetate, treating the resulting product with nitromethane and crystallizing a mixture of ethyl 2,6-didesoXy-2-acetamido-6nitro-a-D-glucothiofuranoside and ethyl 2,6-didesoxy-2-acetamido-6- nitro-B-L-idothiofuranoside.

4. Process which comprises reacting N-acetyl- D-glucosamine with ethyl mercaptan to obtain N.-acetyl-D-glucosamine diethyl thioacetal, reacting the N-acetyl-D-gluccsamine diethyl thioacetal with mercuric chloride and mercuric oxide to obtain ethyl 2-desoxy-2-acetamido-a-D-glucothiofuranoside.

5. Process which comprises reacting N-acetyl- D-glucosamine with ethyl mercaptan to obtain N-acetyl-D-glucosamine diethyl thioacetal,

6. The process of reacting N-acetyl-D-glucosamine diethyl thioacetal with mercuric chloride and mercuric oxide to obtain ethyl Z-desoxy-Z- acetarnido-a-D-glucothiofuranoside.

7. The process of reacting ethyl 2-desoxy-2- acetamido-a-D-glucothiofuranoside with lead tetraacetate, treating the resulting product with nitromethane and crystallizing a, mixture of ethyl 2,6-didesoxy-2-acetamiclo-6-nitro-a-D-glucothiofuranoside and ethyl 2,6-didesoxy-2-acetamido- G-nitro-fi-L-idothiofuranoside.

8. N-acetyl-D-glucosamine diethyl thioacetal.

9. Ethyl 2-desoxy-2-acetamido-a-D-glucothiofuranoside.

10. Ethyl 2,6-didesoxy-2-acetamido-6-nitro-a D-glucothiofuranoside.

l1. Ethyl 2,6-didesoxy-2-acetamido-6-nitro-B- L-idothiofuranoside.

12. Pentaacetyl D-glucosamine diethyl thioacetal.

13. A compound of the class consisting of N-acetyl-D-glucosamine diethyl thioacetal, ethyl 2-desexy-2-acetamido-c-D-glucothiofuranoside, ethyl 2,6-didesoxy-2-acetamido-6-nitro-a-D glucothiofuranoside, ethyl 2,6-didesoXy-2-acetamido-6-nitro-B-L-idothiofuranoside and pentaacetyl D-glucosamine diethyl thioacetal.

MELVILLE LAWRENCE WOLFROM. STEPHEN MUNRO OLIN.

REFERENCES CITED The following references are of record in the file of this patent:

Peck: Jour. Am. Chem. Soc, 68, 779 (1946). Carter: Science, 103, 53-54 (1946). 

1. PROCESS OF PREPARING STREPTAMINE WHICH COMPRISES REACTING N-ACETYL-D-GLUCOSAMINE WITH ETHYL MERCAPTAN TO OBTAIN N-ACETYL-D-GLUCOSAMINE DIETHYL THIOACETAL, REACTING THE N-ACETYL-D-GLUCOSAMINE DIETHYL THIOACETAL WITH MERCURIC CHLORIDE AND MERCURIC OXIDE TO OBTAIN ETHYL 2-DESOXY2-ACETAMIDE - A - D-GLUCOTHIOFURANOSIDE, REACTING THE ETHYL 2-DESOXY-2-ACETAMIDO-A-D-GLUCOTHIOFURANOSIDE WITH LEAD TETRAACETATE, TREATING THE RESULTING PRODUCT WITH NITROMETHANE AND CRYSTALLIZING A MIXTURE OF ETHYL 2,6-DIDESOXY-2-ACETAMIDO6-NITRO-A-D-GLUCOTHIOFURANOSIDE AND ETHYL 2,6DIDESOXY-2-ACETAMIDO-6-NITRO-B-L- IDOTHIOFURANOSIDE, SEPARATING, AND DEMERCAPTOLATING THE HIGHER MELTING FORM, INTRAMOLECURLARLY CONDENSING, HYDROGENATING AND ACETYLATING TO STREPTAMINE HEXAACETATE, WHICH IS SUBSEQUENTLY HYDROLYZED TO STREPTAMINE. 